Structure–Activity Relationship Analysis of Rhosin, a RhoA GTPase Inhibitor, Reveals a New Class of Antiplatelet Agents

Current antiplatelet therapies have several clinical complications and are mostly irreversible in terms of suppressing platelet activity; hence, there is a need to develop improved therapeutic agents. Previous studies have implicated RhoA in platelet activation. Here, we further characterized the lead RhoA inhibitor, Rhosin/G04, in platelet function and present structure–activity relationship (SAR) analysis. A screening for Rhosin/G04 analogs in our chemical library by similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. A screening for Rhosin/G04 analogs in our chemical library using similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. SAR analysis revealed that the active compounds have a quinoline group optimally attached to the hydrazine at the 4-position and halogen substituents at the 7- or 8-position. Having indole, methylphenyl, or dichloro-phenyl substituents led to better potency. Rhosin/G04 contains a pair of enantiomers, and S-G04 is significantly more potent than R-G04 in inhibiting RhoA activation and platelet aggregation. Furthermore, the inhibitory effect is reversible, and S-G04 is capable of inhibiting diverse-agonist-stimulated platelet activation. This study identified a new generation of small-molecule RhoA inhibitors, including an enantiomer capable of broadly and reversibly modulating platelet activity.


Introduction
Platelets are small, anucleated cellular fragments released by bone marrow precursors, megakaryocytes. The primary role of platelets is to maintain hemostasis by serving as sentinels of vascular integrity. Platelets encounter the subendothelial extracellular matrix upon vascular damage and undergo adhesion, shape change, granular secretion, and aggregation, resulting in thrombus formation [1]. Highly reactive platelets in diabetes, hypertension, or plaque rupture in atherosclerotic patients contribute to pathologic thrombosis, leading to life-threatening cardiovascular complications such as myocardial infarction and stroke [2][3][4].
Due to the vital role of activated platelets in the development of cardiovascular diseases, antiplatelet agents such as aspirin and clopidogrel are widely prescribed to prevent and manage thrombotic complications in high-risk individuals. Aspirin was the first and

Results and Discussion
Rhosin/G04 was identified by means of the RhoA-targeted docking of more than four million compounds from an in silico compound library obtained with the ZINC purchasable compound set (International Zinc Association, Washington, DC) [23,34]. Rhosin/G04 contains two aromatic rings tethered by a linker that is believed to bind to the Trp58 region of the RhoA surface required for GEF interaction with micromolar affinity [23,24]. Rhosin/G04 can be synthesized by means of the formal condensation of the carboxy group of D-tryptophan with the amino group of (quinoxalin-6-yl)methylidenehydrazide [35]. Based on the findings obtained by Shang et al. [23], we believe that the preliminary pharmacophore consisting of an Indole/Benzimidazole ring tethered to a quinoxaline through a flexible linker of four-eight atoms retains inhibitory activity against RhoA. The University of Cincinnati (UC)/Cincinnati Children's Hospital Medical Center (CCHMC) compound library of over 360,000 compounds was filtered using a series of substructure and similarity searches for compounds broadly related to this pharmacophore. Seven compounds were selected as they are diverse, show plausible binding pose and score, adhere to the preliminary pharmacophore, and avoid problematic functionality (pan-assay interference compounds (PAINSs), reactive/unstable, or toxicology-linked).
Light transmission aggregometry, i.e., the Born method, was used to determine in vitro antiplatelet aggregation activity using collagen as the agonist for platelet aggregation [36].
Collagen was selected as the agonist, since it is the most thrombogenic component of the subendothelial layer [37]. Collagen also serves as a substrate for platelet adhesion and as an inducer of platelet activation [38,39]. The inhibitory potential of these seven compounds was assessed using a low collagen concentration (1 µg/mL) and a high collagen concentration (5 µg/mL), since cyclooxygenase (COX) inhibitors impact low-collagen-induced aggregation but higher concentrations are unaffected [40,41]. Indomethacin (20 µM), a reversible COX-1 inhibitor, and cangrelor (20 µM), a reversible P 2 Y 12 inhibitor, were chosen as the positive controls to compare the anti-aggregation effect of Rhosin analogs [42][43][44]. UC-177629 emerged as the only potent inhibitor of collagen-induced platelet aggregation in washed human platelets (Table 1). Thirteen analogs of UC-177629 were also evaluated in the platelet aggregation assay at low and high concentrations of collagen, with eight of the compounds showing good activity and five compounds showing minimal activity. These analogs and ten additional compounds that are less closely related but have functionality that might improve physical properties were evaluated; however, the activity was not as compelling (Table 2). Therefore, the top 12 most active compounds that inhibited collagen-induced aggregation by at least 70% in washed human platelets were selected for subsequent analysis. Light transmission aggregometry, i.e., the Born method, was used to determine in vitro antiplatelet aggregation activity using collagen as the agonist for platelet aggregation [36]. Collagen was selected as the agonist, since it is the most thrombogenic component of the subendothelial layer [37]. Collagen also serves as a substrate for platelet adhesion and as an inducer of platelet activation [38,39]. The inhibitory potential of these seven compounds was assessed using a low collagen concentration (1 µg/mL) and a high collagen concentration (5 µg/mL), since cyclooxygenase (COX) inhibitors impact low-collagen-induced aggregation but higher concentrations are unaffected [40,41]. Indomethacin (20 µM), a reversible COX-1 inhibitor, and cangrelor (20 µM), a reversible P2Y12 inhibitor, were chosen as the positive controls to compare the anti-aggregation effect of Rhosin analogs [42][43][44]. UC-177629 emerged as the only potent inhibitor of collagen-induced platelet aggregation in washed human platelets (Table 1). Thirteen analogs of UC-177629 were also evaluated in the platelet aggregation assay at low and high concentrations of collagen, with eight of the compounds showing good activity and five compounds showing minimal activity. These analogs and ten additional compounds that are less closely related but have functionality that might improve physical properties were evaluated; however, the activity was not as compelling (Table 2). Therefore, the top 12 most active compounds that inhibited collagen-induced aggregation by at least 70% in washed human platelets were selected for subsequent analysis. Light transmission aggregometry, i.e., the Born method, was used to determine in vitro antiplatelet aggregation activity using collagen as the agonist for platelet aggregation [36]. Collagen was selected as the agonist, since it is the most thrombogenic component of the subendothelial layer [37]. Collagen also serves as a substrate for platelet adhesion and as an inducer of platelet activation [38,39]. The inhibitory potential of these seven compounds was assessed using a low collagen concentration (1 µg/mL) and a high collagen concentration (5 µg/mL), since cyclooxygenase (COX) inhibitors impact low-collagen-induced aggregation but higher concentrations are unaffected [40,41]. Indomethacin (20 µM), a reversible COX-1 inhibitor, and cangrelor (20 µM), a reversible P2Y12 inhibitor, were chosen as the positive controls to compare the anti-aggregation effect of Rhosin analogs [42][43][44]. UC-177629 emerged as the only potent inhibitor of collagen-induced platelet aggregation in washed human platelets (Table 1). Thirteen analogs of UC-177629 were also evaluated in the platelet aggregation assay at low and high concentrations of collagen, with eight of the compounds showing good activity and five compounds showing minimal activity. These analogs and ten additional compounds that are less closely related but have functionality that might improve physical properties were evaluated; however, the activity was not as compelling (Table 2). Therefore, the top 12 most active compounds that inhibited collagen-induced aggregation by at least 70% in washed human platelets were selected for subsequent analysis. Light transmission aggregometry, i.e., the Born method, was used to determine in vitro antiplatelet aggregation activity using collagen as the agonist for platelet aggregation [36]. Collagen was selected as the agonist, since it is the most thrombogenic component of the subendothelial layer [37]. Collagen also serves as a substrate for platelet adhesion and as an inducer of platelet activation [38,39]. The inhibitory potential of these seven compounds was assessed using a low collagen concentration (1 µg/mL) and a high collagen concentration (5 µg/mL), since cyclooxygenase (COX) inhibitors impact low-collagen-induced aggregation but higher concentrations are unaffected [40,41]. Indomethacin (20 µM), a reversible COX-1 inhibitor, and cangrelor (20 µM), a reversible P2Y12 inhibitor, were chosen as the positive controls to compare the anti-aggregation effect of Rhosin analogs [42][43][44]. UC-177629 emerged as the only potent inhibitor of collagen-induced platelet aggregation in washed human platelets (Table 1). Thirteen analogs of UC-177629 were also evaluated in the platelet aggregation assay at low and high concentrations of collagen, with eight of the compounds showing good activity and five compounds showing minimal activity. These analogs and ten additional compounds that are less closely related but have functionality that might improve physical properties were evaluated; however, the activity was not as compelling (Table 2). Therefore, the top 12 most active compounds that inhibited collagen-induced aggregation by at least 70% in washed human platelets were selected for subsequent analysis. Light transmission aggregometry, i.e., the Born method, was used to determine in vitro antiplatelet aggregation activity using collagen as the agonist for platelet aggregation [36]. Collagen was selected as the agonist, since it is the most thrombogenic component of the subendothelial layer [37]. Collagen also serves as a substrate for platelet adhesion and as an inducer of platelet activation [38,39]. The inhibitory potential of these seven compounds was assessed using a low collagen concentration (1 µg/mL) and a high collagen concentration (5 µg/mL), since cyclooxygenase (COX) inhibitors impact low-collagen-induced aggregation but higher concentrations are unaffected [40,41]. Indomethacin (20 µM), a reversible COX-1 inhibitor, and cangrelor (20 µM), a reversible P2Y12 inhibitor, were chosen as the positive controls to compare the anti-aggregation effect of Rhosin analogs [42][43][44]. UC-177629 emerged as the only potent inhibitor of collagen-induced platelet aggregation in washed human platelets (Table 1). Thirteen analogs of UC-177629 were also evaluated in the platelet aggregation assay at low and high concentrations of collagen, with eight of the compounds showing good activity and five compounds showing minimal activity. These analogs and ten additional compounds that are less closely related but have functionality that might improve physical properties were evaluated; however, the activity was not as compelling (Table 2). Therefore, the top 12 most active compounds that inhibited collagen-induced aggregation by at least 70% in washed human platelets were selected for subsequent analysis. Light transmission aggregometry, i.e., the Born method, was used to determine in vitro antiplatelet aggregation activity using collagen as the agonist for platelet aggregation [36]. Collagen was selected as the agonist, since it is the most thrombogenic component of the subendothelial layer [37]. Collagen also serves as a substrate for platelet adhesion and as an inducer of platelet activation [38,39]. The inhibitory potential of these seven compounds was assessed using a low collagen concentration (1 µg/mL) and a high collagen concentration (5 µg/mL), since cyclooxygenase (COX) inhibitors impact low-collagen-induced aggregation but higher concentrations are unaffected [40,41]. Indomethacin (20 µM), a reversible COX-1 inhibitor, and cangrelor (20 µM), a reversible P2Y12 inhibitor, were chosen as the positive controls to compare the anti-aggregation effect of Rhosin analogs [42][43][44]. UC-177629 emerged as the only potent inhibitor of collagen-induced platelet aggregation in washed human platelets (Table 1). Thirteen analogs of UC-177629 were also evaluated in the platelet aggregation assay at low and high concentrations of collagen, with eight of the compounds showing good activity and five compounds showing minimal activity. These analogs and ten additional compounds that are less closely related but have functionality that might improve physical properties were evaluated; however, the activity was not as compelling (Table 2). Therefore, the top 12 most active compounds that inhibited collagen-induced aggregation by at least 70% in washed human platelets were selected for subsequent analysis.              The cytotoxicity of the active analogs was determined with the lactate dehydrogenase (LDH) leakage assay to ensure that platelet inhibition was a consequence of pharmacologic inhibition and not cytotoxicity. LDH is a stable cytosolic marker for membrane perturbation and is present in many cell types, including platelets [45,46]. The results revealed that none of the 12 analogs differed significantly from DMSO (dimethyl sulfoxide)treated platelets (Supporting Information Figure S2). Next, dose-response curves for platelet aggregation using collagen (1 µg/mL) as the agonist were used to determine the 9.7 ± 6 11.4 ± 9.9 UC-177616 The cytotoxicity of the active analogs was determined with the lactate dehydrogenase (LDH) leakage assay to ensure that platelet inhibition was a consequence of pharmacologic inhibition and not cytotoxicity. LDH is a stable cytosolic marker for membrane perturbation and is present in many cell types, including platelets [45,46]. The results revealed that none of the 12 analogs differed significantly from DMSO (dimethyl sulfoxide)treated platelets (Supporting Information Figure S2). Next, dose-response curves for platelet aggregation using collagen (1 µg/mL) as the agonist were used to determine the 11 The cytotoxicity of the active analogs was determined with the lactate dehydrogenase (LDH) leakage assay to ensure that platelet inhibition was a consequence of pharmacologic inhibition and not cytotoxicity. LDH is a stable cytosolic marker for membrane perturbation and is present in many cell types, including platelets [45,46]. The results revealed that none of the 12 analogs differed significantly from DMSO (dimethyl sulfoxide)-treated platelets (Supporting Information Figure S2). Next, dose-response curves for platelet aggregation using collagen (1 µg/mL) as the agonist were used to determine the half-maximal inhibitory concentration (IC 50 ) of the 12 active compounds. The IC 50 and 95% CI (confidence interval) values of the active analogs ranged from 0.8 µM to 5.6 µM ( Figure 1).
The inhibitory potential of the analogs that were not subjected to IC 50 value determination was estimated based on the percent inhibition observed at 20 µM. The SAR within this series was clear and specific, as follows: (1) The quinoline moiety is optimally attached to the hydrazine at the 4-position. (2) Chloro-and trifluoromethyl substituents at the 7-or 8-position improved activity vs. unsubstituted quinolines. (3) Comparable potency was seen in the indole and methylphenyl substituents, with slightly better potency having been observed in the dichloro-phenyl group (UC-177619). (4) Replacement of the quinoline moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency.
To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they confirmed UC-177619; at RT = 3.79, they confirmed UC-177628; at RT = 3.18, they confirmed UC-177629; at RT = 3.14, they confirmed UC-177633; and at RT = 2.68, they confirmed UC-177634. The inhibitory potential of the analogs that were not subjected to IC50 value determination was estimated based on the percent inhibition observed at 20 µM. The SAR within this series was clear and specific, as follows: (1) The quinoline moiety is optimally attached to the hydrazine at the 4-position. (2) Chloro-and trifluoromethyl substituents at the 7-or 8-position improved activity vs. unsubstituted quinolines. (3) Comparable potency was seen in the indole and methylphenyl substituents, with slightly better potency having been observed in the dichloro-phenyl group (UC-177619). (4) Replacement of the quino- Several studies reported that N-acylheteroaryl hydrazones (NAHs) show efficacy as platelet aggregation inhibitors. A study by Chelucci et al. reported that nonsteroidal anti-inflammatory drugs (NSAIDs) containing the NAH moiety might exhibit better antiplatelet and antithrombotic activity than aspirin [27]. Another study showed that the substituent on the phenyl ring of the NAH subunit might be responsible for improved antiplatelet activity in the presence of multiple agonists, such as arachidonic acid (AA), collagen, and adenosine diphosphate (ADP) [47]. Fraga et al. showed that acylhydrazone derivatives had inhibitory effects on platelet-activating factor (PAF) and presented a significant antithrombotic profile [48]. In silico, in vitro, and in vivo investigations conducted by Khalid et al. showed that a series of hydrazone and sulfonamide derivatives inhibited AA-, ADP-, and collagen-induced platelet aggregation [49]. Researchers at LASSBio have published several papers showing that hydrazine and acylhydrazone moieties are the essential pharmacophoric cores for several antiplatelets, NSAIDs, and analgesic derivatives due to their inhibitory effects on COX and/or 5-lipooxygenase (LOX) [26,29,31]. The same group also reported that arylsulfonate-acylhydrazone derivatives could simultaneously inhibit platelet aggregation induced by collagen, AA, and thrombin [50]. Table 3. Structure-activity relationship of UC-177629 analogs derived by arranging the compounds with variations in the quinoline group across the vertical axis and placing compounds with variations in their aryl group across the horizontal axis. line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they IC 50 = 4.59 µM line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they IC 50 > 20 µM line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they IC 50 = 2.84 µM line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they IC 50 = 1.5 µM line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they IC 50 = 2.34 µM line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they IC 50 > 20 µM line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they IC 50 > 20 µM line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they line moiety with an indane (UC-178838) surprisingly afforded a nearly equipotent compound, while a positional isomer (e.g., UC-177630) lost all activity (Table 3). Analogs UC-177618, UC-177619, UC-177628, UC-177629, UC-177633, and UC-177634 were selected for subsequent analysis due to their potency. To confirm the structures of the six most active analogs, they were subjected to ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS was performed to determine the accurate mass-to-charge ratio (m/z), isotope/adduct ion patterns, and fragmentation patterns. No significant degradation product was present, and the results of the six active analogs are shown in Supporting Information Figure S3. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 6.42 min confirmed UC-177618; at RT = 4.82 min, they

Aryl Group
In addition to hydrazone, the Indole ring is also considered an essential structural moiety with antiplatelet activity. Park et al. analyzed the antiplatelet activity of Indole-3-carbinol, an autolysis product of cruciferous vegetables. They found that Indole-3carbinol could be a potential antithrombotic and antiplatelet agent, since it inhibited collagen-induced platelet aggregation [32]. Mashayekhi et al. synthesized a series of indole hydrazine derivatives and tested their anti-aggregation ability. They found that these compounds inhibited ADP-and AA-induced platelet aggregation [30]. Akhlaghi et al. synthesized N-aryl methyl-substituted Indole derivatives and showed that these compounds effectively inhibited ADP-and AA-induced platelet aggregation [51]. Tavili et al. synthesized and evaluated a series of N1-substituted indole hydrazones and found that they inhibited AA-induced platelet aggregation. Their SAR analysis revealed that lipophilicity of the para-substituted phenyl ring contributed to the increase in the antiplatelet activity of the derivatives [33]. Tehrani et al. showed that the compounds that share a strongly preserved structural backbone, two (hetero) aromatic ring systems linked by a hydrazone bond, inhibited aggregation induced by AA and ADP [28]. Based on the evidence from these studies, it is likely that the antiplatelet activity of these individual pharmacophoric components might be related to RhoA inhibition.
Previously, we showed that removing Rhosin by washing reversed its inhibitory effects on NIH 3T3 cells [23]. The reversible inhibition of Rhosin in collagen-induced platelet aggregation was assessed with similar wash-out experiments, and this reversible nature of Rhosin makes it a valuable antiplatelet drug candidate (Supporting Information Figure S4A,B). After confirming the structures of the above six active analogs, similar washout experiments were performed to compare the reversible function of the active analogs with Rhosin reversibility. Supporting Information Figure S4C,D shows the representative aggregation tracings for UC-177618 and UC-177619, confirming that similar to those of Rhosin/G04, the inhibitory effects of these Rhosin analogs are entirely reversible.
Next, the ability of the six compounds to suppress RhoA activity in platelets was assessed by subjecting them to a GST (Glutathione-S-transferase)-Rhotekin effector pulldown assay. The compounds were incubated with washed human platelets for 2 min before being stimulated with 1 µg/mL collagen. The analogs inhibited the activation of RhoA at the 20 µM concentration (Figure 2A,B). The analogs also inhibited the phosphorylation of its downstream effector, the MLC ( Figure 2C,D). After confirming that these analogs could prevent RhoA activation and the phosphorylation of the MLC, their ability to disrupt complex-formation between RhoA and LARG was tested. The analogs at 20 µM suppressed LARG binding to RhoA under pull-down assay conditions, while inactive analog UC-177626 showed no effects ( Figure 3A). Docking analysis (based on PDB ID 5C4M) was used to demonstrate the binding of the top two active analogs, UC-177618 and UC-177619, to RhoA to show that the quinolines of these compounds formed pi-stacking with the Trp58 residue ( Figure 3B). These findings indicate that the active analogs inhibit RhoA activation by disrupting the binding between RhoA and its GEF. UC-177626 showed no effects ( Figure 3A). Docking analysis (based on PDB ID 5C4M) was used to demonstrate the binding of the top two active analogs, UC-177618 and UC-177619, to RhoA to show that the quinolines of these compounds formed pi-stacking with the Trp58 residue ( Figure 3B). These findings indicate that the active analogs inhibit RhoA activation by disrupting the binding between RhoA and its GEF.  Rhosin/G04 is probably derived from R-tryptophan with E geometry across the C=N bond, i.e., Rhosin is the R enantiomer (R-G04). The natural tryptophan enantiomer of G04, i.e., S-G04, was also evaluated. The cytotoxicity of both R-G04 and S-G04 at 50 µM was evaluated in washed human platelets using the LDH assay. As shown in Supporting Information Figure S5, neither R-G04 nor S-G04 induced significant human platelet cytolysis compared with the positive control, membrane detergent Triton X-100. After assessing the non-cytotoxicity of these enantiomers, UHPLC-HRMS was performed, and no significant degradation products were observed. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 1.83 min confirmed R-G04, while the highresolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 1.85 min confirmed S-G04 (Supporting Information Figure S6).
S-G04 appeared to be better at inhibiting collagen-induced platelet aggregation than the standard drugs (Table 4). Hence, further antiplatelet analysis of G04 stereoisomers was performed using concentration-response curves using collagen (1 µg/mL) as the agonist. The collagen-induced platelet aggregation assay showed that the IC50 value of S- Rhosin/G04 is probably derived from R-tryptophan with E geometry across the C=N bond, i.e., Rhosin is the R enantiomer (R-G04). The natural tryptophan enantiomer of G04, i.e., S-G04, was also evaluated. The cytotoxicity of both R-G04 and S-G04 at 50 µM was evaluated in washed human platelets using the LDH assay. As shown in Supporting Information Figure S5, neither R-G04 nor S-G04 induced significant human platelet cytolysis compared with the positive control, membrane detergent Triton X-100. After assessing the non-cytotoxicity of these enantiomers, UHPLC-HRMS was performed, and no significant degradation products were observed. The high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 1.83 min confirmed R-G04, while the high-resolution mass spectrum of the peak and the fragmentation pattern of the peak at RT = 1.85 min confirmed S-G04 (Supporting Information Figure S6).
S-G04 appeared to be better at inhibiting collagen-induced platelet aggregation than the standard drugs (Table 4). Hence, further antiplatelet analysis of G04 stereoisomers was performed using concentration-response curves using collagen (1 µg/mL) as the agonist. The collagen-induced platelet aggregation assay showed that the IC 50 value of S-G04 is 7.80 µM, which makes it three times more potent than R-G04 (Figure 4). To examine the specificity of R-G04 and S-G04 in suppressing RhoA activity in platelets, platelets were pre-incubated with different concentrations of R-G04 or S-G04 before being subjected to the GST-Rhotekin effector pull-down assay. As shown in Figure 5B, both R-G04 and S-G04 inhibited RhoA-GTP formation in a concentration-dependent manner. Furthermore, both enantiomers of G04 also inhibited the downstream phosphorylation of the MLC, which is mediated by RhoA effector ROCK ( Figure 5C,D). Our findings are further corroborated by Shang et al. [23] and Huzoor et al. [25], who also showed that Rhosin/G04 specifically inhibits the activation of RhoA but not that of Rac1 and Cdc42. which is mediated by RhoA effector ROCK ( Figure 5C,D). Our findings are further corroborated by Shang et al. [23] and Huzoor et al. [25], who also showed that Rhosin/G04 specifically inhibits the activation of RhoA but not that of Rac1 and Cdc42.   which is mediated by RhoA effector ROCK ( Figure 5C,D). Our findings are further corroborated by Shang et al. [23] and Huzoor et al. [25], who also showed that Rhosin/G04 specifically inhibits the activation of RhoA but not that of Rac1 and Cdc42. which is mediated by RhoA effector ROCK ( Figure 5C,D). Our findings are further corroborated by Shang et al. [23] and Huzoor et al. [25], who also showed that Rhosin/G04 specifically inhibits the activation of RhoA but not that of Rac1 and Cdc42.    To confirm the mechanism of inhibition of G04 enantiomers, a complex-formation assay with RhoA and the LARG DH-PH module was performed by incubating them with RhoA. Both R-G04 and S-G04 were capable of dose-dependently suppressing LARG binding to RhoA under pull-down assay conditions ( Figure 6A). Figure 6B shows a hypothetical docking model of R-G04 (cyan) and S-G04 (magenta) in RhoA. RhoA is a flexible protein, particularly across the Switch 1 and Switch 2 regions, which are involved in G04 binding and GEF interaction [23,52]. Docking studies (based on PDB ID 5C4M) were used to depict that the R and S enantiomers of G04 bind similarly, with the quinoxaline anchored with a pi-stacking interaction to Trp-58 within a lipophilic pocket and the indole across Leu-69 and Leu-72, respectively. The amine group of R-G04 formed a hydrogen bond with the Glu-40 residue (3.1 Å), while the amine of S-G04 formed a hydrogen bond with the Ser-73 residue (2.9 Å), which may account for the relatively small three-fold difference in the potency of these compounds. These findings are consistent with our previous work, which found that Rhosin/R-G04 fails to bind to RhoA point mutants bearing Ala mutations around the GEF recognition site [23]. To confirm the mechanism of inhibition of G04 enantiomers, a complex-formation assay with RhoA and the LARG DH-PH module was performed by incubating them with RhoA. Both R-G04 and S-G04 were capable of dose-dependently suppressing LARG binding to RhoA under pull-down assay conditions ( Figure 6A). Figure 6B shows a hypothetical docking model of R-G04 (cyan) and S-G04 (magenta) in RhoA. RhoA is a flexible protein, particularly across the Switch 1 and Switch 2 regions, which are involved in G04 binding and GEF interaction [23,52]. Docking studies (based on PDB ID 5C4M) were used to depict that the R and S enantiomers of G04 bind similarly, with the quinoxaline anchored with a pi-stacking interaction to Trp-58 within a lipophilic pocket and the indole across Leu-69 and Leu-72, respectively. The amine group of R-G04 formed a hydrogen bond with the Glu-40 residue (3.1 Å), while the amine of S-G04 formed a hydrogen bond with the Ser-73 residue (2.9 Å), which may account for the relatively small three-fold difference in the potency of these compounds. These findings are consistent with our previous work, which found that Rhosin/R-G04 fails to bind to RhoA point mutants bearing Ala mutations around the GEF recognition site [23]. After identifying an enantiomer with significantly higher potency than the original R-G04, we applied the enantiomers to antiplatelet assays for inhibiting platelet aggregation induced by multiple agonists, including AA, ADP, U46619, a stable synthetic analog of thromboxane A2 (TxA2), and thrombin. As shown in Figure 7A-D, 10 µM S-G04 suppressed the platelet activation induced by AA, U46619, and thrombin, and it also inhibited ATP secretion when stimulated with thrombin. Pre-incubating platelets with 20 µM R-G04 failed to have the same inhibitory effect. However, both R-G04 (20 µM) and S-G04 (10 µM) significantly inhibited ADP-induced platelet aggregation. In addition, R-G04 and S-G04 exhibited dose-dependent inhibition on the generation of TxA2, measured by means of its stable metabolite, thromboxane B2 (TxB2). TxA2 is a metabolite derived from AA via the COX pathway, a potent vasoconstrictor, and a platelet agonist that results in irreversible platelet aggregation [53][54][55]. The inhibitory potential of S-G04 was comparable to that of Indomethacin, a COX inhibitor ( Figure 7F). The data in Figure 7 show the potential of the RhoA inhibitors to inhibit platelet aggregation mediated by diverse agonists, i.e., TxA2, ADP, U46619, AA, and thrombin, and ATP secretion, highlighting the potential to inhibit multiple-agonist-mediated platelet activation pathways at micromolar concentrations. After identifying an enantiomer with significantly higher potency than the original R-G04, we applied the enantiomers to antiplatelet assays for inhibiting platelet aggregation induced by multiple agonists, including AA, ADP, U46619, a stable synthetic analog of thromboxane A 2 (TxA 2 ), and thrombin. As shown in Figure 7A-D, 10 µM S-G04 suppressed the platelet activation induced by AA, U46619, and thrombin, and it also inhibited ATP secretion when stimulated with thrombin. Pre-incubating platelets with 20 µM R-G04 failed to have the same inhibitory effect. However, both R-G04 (20 µM) and S-G04 (10 µM) significantly inhibited ADP-induced platelet aggregation. In addition, R-G04 and S-G04 exhibited dose-dependent inhibition on the generation of TxA 2 , measured by means of its stable metabolite, thromboxane B 2 (TxB 2 ). TxA 2 is a metabolite derived from AA via the COX pathway, a potent vasoconstrictor, and a platelet agonist that results in irreversible platelet aggregation [53][54][55]. The inhibitory potential of S-G04 was comparable to that of Indomethacin, a COX inhibitor ( Figure 7F). The data in Figure 7 show the potential of the RhoA inhibitors to inhibit platelet aggregation mediated by diverse agonists, i.e., TxA2, ADP, U46619, AA, and thrombin, and ATP secretion, highlighting the potential to inhibit multiple-agonist-mediated platelet activation pathways at micromolar concentrations. Washed human platelets were pre-incubated with 10 µM S-G04 or 20 µM R-G04 for 2 min before being stimulated with Arachidonic Acid (AA), U46619, ADP, or Thrombin. The % platelet aggregation was measured 5 min after agonist stimulation. (E) Washed human platelets were pre-incubated with 10 µM S-G04 or 20 µM R-G04 for 2 min and with Chrono-lume ® for 30 s at 37 °C before being stimulated with 0.1 U/mL Thrombin. (F) Washed human platelets were pre-incubated with different concentrations of R-G04 or S-G04 or with 20 µM Indomethacin for 2 min before being stimulated with Thrombin. TxB2 formation was determined using a TxB2 ELISA kit. The differences among DMSO, R-G04, and S-G04 were analyzed using t-test, and the results are shown as means ± SDs of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Virtual Screening
The UC/CCHMC compound library of over 360,000 chemicals was scanned for Rhosin analogs using similarity and substructure searches. These cheminformatics searches were performed in Pipeline Pilot (version 18.1.100.11; Dassault Systemes BioVia). Similarity searches were based on ECFP4, FCFP4, and FCFC4 fingerprints using Tanimoto similarity calculations. This library was subjected to a virtual screening of the GDP/GTP binding pocket and the GEF binding site of RhoA. Virtual screening was performed in the ICM-Pro software suite (version 3.8-7; Molsoft LLC) against RhoA structures PDB ID 1Xx86 [52] and 1ftn [56] obtained from Protein Data Bank (available online at www.rcsb.org (accessed on 22 August 2022)) [57], and later dockings included 5C4M [58]. Individual PDB files (PDBIDs 1 × 86, 1ftn) were read in ICM-Pro and stripped of crystallographic artifacts and waters. The docking sites were defined by means of visual inspection in Pymol and verified with the Pocket-Finder function in MolSoft ICM-Pro. The above SD files were read in ICM-Pro and converted into 3D structures (50 low-energy conformers), and H atoms and Gasteiger charges were added; then, the structures were docked at thoroughness of 30.

Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry Analysis (UHPLC-HRMS)
Compounds in DMSO solution (1 mM) were further dissolved into acetonitrile/water (1/1 v/v) to the appropriate concentration for mass spectrometry. Volumes of 10 µL of prepared solution of S-GO4 and R-GO4, and 5 µL of analogs UC-177618 and UC-177619 were injected into a Q ExactiveTM plus hybrid quadrupole-OrbitrapTM mass spectrometer interfaced with a Vanquish ultra-high-performance liquid chromatography (UHPLC) system (Thermo Scientific, Waltham, MA, USA). Chromatography separation was conducted using an Acquity BEH C18 UPLC column (2.1 × 100 mm; 1.7 µm) with a guard column (Waters). The column temperature was set to 20 • C. An isocratic mobile phase was used with a binary solvent system, and 65% solvent A (water with 2 mM ammonium acetate and 0.1% formic acid) and 35% solvent B (acetonitrile) were used in the analysis. The total run time was 10 min, and the flow rate was 0.2 mL/min. The ESI source was operated with the following parameters: spray voltage, 3.4 kV; capillary temperature, 350 • C; sheath gas flow rate, 35; auxiliary gas heater temperature, 325 • C. Data were acquired using a full MS scan (mass scan range of 150-900 m/z, AGC target of 3e6, maximum IT of 100 ms, and resolution of 70,000), and collision-induced dissociation-based data were dependent on MS/MS (resolution of 17,500, AGC target of 1e5, maximum IT of 50 ms, loop count of 7, top N = 7, isolation window of 1.0 m/z, and stepped NCE of 25). Samples were also checked with an additional gradient LC method to confirm the results. Data were analyzed with Xcalibur™ data acquisition and interpretation software and FreeStyle™ 1.6 (Thermo Scientific).

Collection of Blood and Preparation of Washed Human Platelet Suspensions
All experiments using human blood from healthy volunteers were performed according to the protocols approved by the Institutional Review Board at Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. Each volunteer was required to sign an informed consent form approved by the IRB. Procedures for drawing human blood, isolating platelet-rich plasma (PRP), and preparing washed platelet suspensions were the same as those previously reported [59,60]. Blood from healthy human donors who reported having been free of medication for ten days was collected into ACD (2.5% trisodium citrate, 2% dextrose, and 1.5% citric acid) at 1:6 (v/v). PRP was obtained by means of the centrifugation of blood at 1400 rpm for 15 min at room temperature. PRP was centrifuged at 4000 rpm for 10 min at room temperature, and the platelet pellet was resuspended in HEPES Tyrode's solution (pH 7.4) without calcium. Platelets were counted using Hemavet 950FS (Drew Scientific, Oxford, CT, USA), and the count was adjusted to 2.5 × 10 8 /mL for aggregation studies.

Lactate Dehydrogenase (LDH)-Based Cytotoxicity Assay
Compound cytotoxicity was assessed using LDH Cytotoxicity Assay Kit (No. 601170; Cayman Chemical, MI) as previously described [61]. Washed human platelets (2.5 × 10 8 /mL) were incubated at 37 • C for 10 min with the highest concentration of compounds (50 µM for R-G04 and S-G04, and 20 µM for Rhosin analogs) or the vehicle, dimethyl sulfoxide (DMSO) 0.1% or 10% Triton X-100, which served as the positive control. The platelets were centrifuged at 800× g for 8 min at 4 • C, and 100 µL of the supernatant was mixed with 100 µL of LDH reaction solution in a 96-well plate. The plate was incubated at 37 • C for 10 min under constant orbital shaking before measurement of the absorbance at 490 nm in a plate reader (BioTek Synergy LX multimode reader; Santa Clara, CA, USA).

Antiplatelet Aggregation Activity and Secretion of ATP
The in vitro antiplatelet activity of the compounds was assayed on washed human platelets using light transmission aggregometry [36]. Platelet aggregation was performed using a 4-channel Lumi-aggregometer (Chronolog, PA, USA) at 37 • C and stirring speed of 900 rpm [14]. A volume of 250 µL of washed human platelets (2.5 × 10 8 /mL) was prewarmed to 37 • C along with the compounds or DMSO (0.1%) for 2 min before the addition of agonists such as collagen, AA, U46619 (a stable synthetic analog of thromboxane A 2 ), ADP, and thrombin. For ATP secretion, platelets were pre-incubated with a luciferin/luciferase kit (Chrono-lume ® ) for 30 s at 37 • C before the addition of the agonist [60]. The reactions were allowed to proceed for at least 4 min, and the results of platelet aggregation and ATP secretion were determined using Aggrolink software. Platelet aggregation was expressed as the maximum change in the light transmission, where the 100% value corresponded to treatment with the agonist alone. Compounds were evaluated in triplicate at nine compound concentrations ranging from 50 µM to 1 µM. The half-maximal inhibitory concentration (IC 50 ) value was calculated as the concentration of the inhibitor causing 50% inhibition of aggregation.

RhoA GTPase Assay and Phosphorylation of MLC
The relative levels of RhoA-GTP in washed human platelets were quantified with effector domains GST-Rhotekin pull-down assays as previously reported [23]. A volume of 250 µL of washed human platelets (2.5 × 10 8 /mL) was pre-warmed to 37 • C along with the compounds or DMSO (0.1%) for 2 min before being stimulated with collagen (1 µg/mL). The reactions were terminated 4 min after collagen stimulation by adding ice-cold lysis buffer (20 mM Tris·HCl (pH 7.6), 100 mM NaCl, 1% Triton X-100, 0.2% Sodium Deoxycholate, 10 mM MgCl2, 1 mM dithiothreitol, 1x protease inhibitor cocktail, and 1x phosphatase inhibitor cocktail). The supernatants were collected and subjected to the GST-Rhotekin pull-down assay. The total cell lysates were also blotted in parallel. GTP-bound RhoA was quantitatively detected with Western blotting using an anti-RhoA antibody. The relative amounts of RhoA were quantified with densitometry measurements and normalized to the untreated platelets.
A volume of 250 µL of washed human platelets (2.5 × 10 8 /mL) was pre-warmed to 37 • C along with the compounds or DMSO (0.1%) for 2 min before being stimulated with collagen (1 µg/mL) for 4 min. The reactions were terminated by adding 4x Laemmli buffer, and phosphorylated MLC protein was detected with Western blotting as previously reported [15]. Phosphorylation was quantified by measuring the densitometry.

In Vitro Complex-Formation Assay
In total, 100 ng of purified MBP (Maltose-binding protein)-(His)6-tagged LARG DH-PH was immobilized on 20 µL of suspended amylose beads (No. E8021S; New England Biolabs Inc., Ipswich, MA, USA) by incubating them in an orbital shaker at 4 • C for 30 min in binding buffer (20 mM Tris·HCl (pH 7.6), 100 mM NaCl, 0.01% Triton X-100, 5mM EDTA, and 5 mM MgCl 2 ). The compounds were added to the binding buffer containing 100 ng of purified full-length RhoA and pre-incubated at 4 • C for 30 min under constant agitation.
The Amylose beads bound to LARG were washed thrice with binding buffer to get rid of unbound LARG, and the pre-incubated compound-RhoA mixture was added to the beads. This LARG-compound-RhoA mixture was incubated in the orbital shaker at 4 • C for 1 h. The beads were washed three times, and the amount of RhoA protein co-precipitated with the MBP-fusion-bound beads was detected with anti-RhoA Western blotting.

Thromboxane B2 ELISA Assay
The level of TxB 2 was measured with an enzyme immunoassay kit (No. 501020; Cayman Chemical, Ann Arbor, MI, USA) as previously described [32]. A volume of 250 µL of washed human platelets (2.5 × 10 8 /mL) was pre-warmed to 37 • C along with the Rhosin-analogs, Indomethacin, or DMSO (0.1%) for 2 min before being stimulated with thrombin (0.1 U/mL) for 4 min. The reactions were terminated by adding 250 µL of HEPES Tyrode's buffer (pH 7.4) containing 2 mM EDTA. The platelets were centrifuged for 2 min, and the level of TxB 2 in the supernatant was measured with the ELISA kit according to the manufacturer's instructions.

Statistical Analysis
Data were obtained from experiments on samples from at least three healthy volunteers and expressed as means ± standard deviations (SDs) where applicable. All experimental data were analyzed using Prism 9.0 software/Graph Pad Inc., San Diego, CA, USA and compared for statistically significant differences using two-tailed Student's t-test.

Conclusions and Future Directions
We evaluated G04 stereoisomers and a series of closely related compounds in a platelet activity screening to identify a new generation of small-molecule RhoA inhibitors. We determined that the S enantiomer of G04 has significantly higher potency than the initial lead Rhosin/R-G04, and we identified a series of analogs with even higher potency. Based on our SAR analysis, we conclude that an ideal RhoA inhibitor must have a quinoline attached to the hydrazine at the 4-position and that having a halogen substitution at the 7or 8-position is beneficial. Comparable potency was seen in the indole and methylphenyl substituents, with slightly better potency having been observed in the dicholorphenyl group. The replacement of the quinoline moiety with an indane afforded a nearly equipotent compound, while a positional isomer lost all activity. The G04 stereoisomers and the active analogs exhibited a similar mechanism of inhibition of RhoA, whereby they inhibited the binding of LARG to RhoA in the complex-formation assay. Additional platelet activation assays indicated that this class of RhoA inhibitors possesses broad antiplatelet properties.
Although this work focused on testing the antiplatelet activity of these compounds, the lack of knowledge about their biodisponibility, pharmacokinetics, and pharmacodynamics is a limit to their pharmacological advancement. This limitation can be addressed using in vivo systems and animal models. We realize that the study lacks a deeper understanding of the mechanism of action of RhoA inhibitors; this could be addressed in the future using nuclear magnetic resonance or crystallography (NMR/crystallography) and additional biophysical and biochemical assays. Due to the reversible nature of these inhibitors and their ability to inhibit multiple-agonist-mediated platelet aggregation, they have the potential to be developed into broad and reversible antiplatelet alternative agents to the available antiplatelet drugs on the market, adding to the currently available indomethacin and cangrelor, which work according to distinct mechanisms. Further studies are necessary to understand the pharmacological profile of these RhoA inhibitors and to help them further advance into clinical trials. Funding: This work was partially supported by NIH grants U54 DK126108, R01 CA204895, P01 HL158688, and R01 HL147536.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Cincinnati Children's Hospital Research Foundation (protocol No. 2010-1855; date of approval: 8/4/2010) for studies involving humans.